The Growth and Mechanical Properties of Living Neurons Measured via Atomic Force and Fluorescence Microscopy
Abstract
In this thesis we explore specific properties of the cytoskeleton and growth of living neurons via atomic force and fluorescence microscopies. We make the first comparative elastic modulus measurements on three types of neuronal cells plated on three types of substrate adhesion factors. We discover that during phases of active neurite extension the soma of cortical neurons stiffens reversibly due to changes in microtubule aggregation. Additionally, we demonstrate that mechanical properties of cortical neurons measured near physiological temperatures are primarily dependent on the microtubule component of the cytoskeleton. We further explore the response of the neuronal cytoskeleton to changes in ambient temperature. The elastic modulus of cortical neuron somas is discovered to increase dramatically upon a drop in ambient temperature. We determine through fluorescent staining and chemical modification of the cytoskeleton that this stiffening is due primarily to a change in the mechanically dominant component of the cytoskeleton from microtubules at 37ºC to actin at 25ºC precipitated by changes in myosin II dynamics within the cell. We make the first direct mechanical measurements of the pericellular brush layer on living neurons, demonstrating that the traditionally observed viscoelastic behavior of the neuronal soma is due to the properties of this brush layer. When the brush layer is excluded, the underlying soma is discovered to be both stiffer than previously observed, and elastic, with no loading-speed dependence to the elastic modulus under the test conditions. We additionally demonstrate that the soma elastic modulus, brush length, and brush density are all dependent on the ambient temperature. Finally, through fluorescent and bright field microscopies we track the outgrowth of living neurons on patterned directional surfaces, demonstrating that asymmetrical ratchet topographies unidirectionally bias axonal outgrowth. We model the outgrowth of the neurons using Fokker-Planck formalism, describing an effective torque which aligns the growth cones with the surface ratchet direction. The asymmetry of outgrowth in the ratchet direction is shown to correlate directly to the ratio of angles that the ratchets make with respect to the underlying surface. We further determine that this topographical detection requires normal functioning of both the microtubule and actin/myosin components of the cytoskeleton.
- Publication:
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Ph.D. Thesis
- Pub Date:
- 2014
- Bibcode:
- 2014PhDT.......284S
- Keywords:
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- Physics, Molecular;Physics, Atomic;Biophysics, General